Here we report that the

Here we report that the Tim-3-galectin-9 autocrine loop is activated in AML order RG7388 through protein kinase C (PKC)/mTOR pathways. These pathways trigger translation of both Tim-3 and galectin-9 and induce high levels of galectin-9 secretion as well as the release of soluble Tim-3. Importantly, this effect was also verified in the AML patients studied. Galectin-9 was found to impair AML cell killing by primary human NK cells. Soluble Tim-3 reduced the ability of T cells to secrete IL-2, a cytokine, which is required for the activation of both NK cells and cytotoxic T cells (Dhupkar and Gordon, 2017). Blood plasmas of AML patients contained significantly lower amounts of IL-2 compared to those of healthy donors. We confirmed that PKC activation occurred in AML cells in a LPHN1-dependent manner. The LPHN1 agonist LTX and natural ligand FLRT3 upregulated the Tim-3-galectin-9 autocrine loop in a PKC-dependent manner. Based on our findings, we conclude that LPHN1/PKC/mTOR/Tim-3-galectin-9 is a biosynthetic and secretory pathway which is operated by human AML cells resulting in a decrease of immune surveillance and promotion of disease progression.
Materials and Methods
Results
Discussion AML is a malignancy affecting bone marrow and blood and is a severe, and often fatal, systemic disease. AML cells escape host immune attack involving NK and cytotoxic T cells by impairing their activity (Golden-Mason et al., 2013; Kikushige et al., 2015; Gonçalves Silva et al., 2016). However, the biochemical mechanisms underlying the immune escape of malignant white blood cells remain unclear. Recently, it was shown that AML cells express high levels of the immune receptor Tim-3 and release galectin-9 which impairs the activity of NK cells and cytotoxic T cells (Gonçalves Silva et al., 2016). We have also suggested that Tim-3, as a membrane associated glycoprotein, might act as a trafficker for galectin-9 (Gonçalves Silva et al., 2016). As for all galectins, galectin-9 is synthesized on free ribosomes and since it lacks the signal domain required for secretion it thus needs a trafficker in order to be released (Delacour et al., 2009). When on the cell surface, Tim-3 is known to be shed by ADAM 10/17 proteolytic enzymes thus producing sTim-3, the function of which remains unknown (Moller-Hackbarth et al., 2013). We found, that Tim-3 could be shed in its free form as well as in complex with galectin-9; however, differential shedding is taking place. The Tim-3 fragment in the complex is about 20kDa molecular weight, while sTim-3 is around 33kDa. SRCD analysis of the complex suggests that the interaction between Tim-3 and galectin-9 proteins leads to major conformational change, possibly increasing the ability of galectin-9 to interact with the target proteins. Since galectin-9 is a tandem protein containing two domains (Delacour et al., 2009), one of them might be interacting with Tim-3, while the other one could bind to a target receptor molecule, for example another molecule of Tim-3 associated with the plasma membrane of the target cell (Nagae et al., 2006). This may explain the high efficiency of galectin-9 in triggering Tim-3 on NK cells, which do not express galectin-9 and thus contain unoccupied Tim-3 on their surface. Since we can observe the Tim-3-galectin-9 complex on Western blots following denaturing SDS-gel electrophoresis (Figs. 1, 2 and 4; ~52kDa soluble form and ~70kDa cell-derived form), it is likely that the binding between proteins is further strengthened by the interaction of galectin-9 with Tim-3-associated glycosides. Interestingly, the complex is detectable by Western blot with both anti-Tim-3 and anti-galectin-9 antibodies. However, when these antibodies are sequentially applied to the same blot, the second antibody fails to detect the respective protein in the same band (unless the first antibody is stripped off), due to steric hindrance. This effect explains why Tim-3 located on the cell surface and covered by galectin-9 cannot be co-stained by the antibody in confocal microscopy co-localization analysis (Fig. 3). Another point supporting this conclusion is that there was also clear evidence of co-localization of Tim-3 and galectin-9 in permiabilized THP-1 cells upon exposure to PMA (Fig. 3, Supplementary Fig. 1).